Effects of a Pure Α/Β-Adrenergic Receptor Blocker on Monocrotaline-Induced Pulmonary Arterial Hypertension with Right Ventricular Hypertrophy in Rats

Advance Publication by J-STAGE

Effects of a Pure α/β-Adrenergic Receptor Blocker on Monocrotaline-Induced Pulmonary Arterial Hypertension With Right Ventricular Hypertrophy in Rats

Masaya Ishikawa, MD; Naoki Sato, MD*; Kuniya Asai, MD; Teruo Takano, MD; Kyoichi Mizuno, MD

Background: It is unclear how much the sympathetic nervous system is involved in the development of pulmonary arterial hypertension (PAH). The present study examined whether or not a pure α/β-adrenergic receptor blocker (arotinolol) could prevent the development of PAH and right ventricular hypertrophy (RVH) in a rat model of monocrotaline (MCT)-induced PAH. Methods and Results: The heart rate, arterial blood pressure (BP), left ventricular pressure, pulmonary artery pressure (PAP), and right ventricular pressure (RVP) were measured after administration of arotinolol or saline for 2 weeks. Ventricular weight and myocyte size were also measured. Mean PAP was increased less in the arotinolol group (n=6), (53±9 vs 21±2 mmHg in the control (n=6); P<0.01). Systolic RVP was also less in the arotinolol group (41±3 vs 91±14 mmHg in the control, P<0.05) without differences in BP. It also significantly reduced the RV/body weight ratio (0.58±0.01 vs 0.77±0.04 mg/g; P<0.01). Furthermore, the myocyte width was significantly decreased in the arotinolol group. Conclusions: The pure α/β-blocker arotinolol prevented the progression of MCT-induced PAH and RVH in rats, suggesting that sympathetic nervous activation might play a role in the development of PAH.

Key Words: α /β-adrenergic receptor blocker; Pulmonary arterial hypertension; Sympathetic nervous system

ulmonary arterial hypertension (PAH) is a serious chronic disorder, similar to chronic kidney disease, Editorial p ???? P which has varied causes and pathogenesis. PAH can potentially lead to right ventricular hypertrophy (RVH) Therefore, we investigated whether or not arotinolol, and cardiovascular death related to RV failure. Among which differs from carvedilol with regard to actions such as the types of PAH, idiopathic PAH has an especially poor an antioxidant or calcium-channel blocking effect,4,5 could prognosis and is difficult to manage clinically. Idiopathic prevent the development of PAH and/or RVH in a rat model PAH involves multiple factors, such as vasoconstriction, of monocrotaline (MCT)-induced PAH. remodeling of the pulmonary vessels, and thrombosis, but it is unclear to what extent the sympathetic nervous activation plays a role in its development. Certainly, sympathetic Methods nervous activity is reported to be increased in PAH;1 The experimental procedures described in the present study however, it is unclear whether or not inhibition of sympa- were approved by the institutional Animal Care and Use thetic activity can prevent or reverse PAH. The benefits of Committee of Nippon Medical School. Six-week-old male inhibiting sympathetic nervous activation in PAH remain Wistar rats (n=12) were used. MCT was dissolved in 1 N uncertain, although there have been several reports regard- HCl, and the pH was adjusted to 7.4 with 1 N NaOH. Rats ing the effects of α- and/or β-adrenergic receptor blockers, were injected subcutaneously with 2% MCT solution at a especially carvedilol, although it is not a pureα /β-adrenergic dose of 40 mg/kg. An intraperitoneal osmotic pump (Alzet receptor blocker. Arotinolol (Dainippon Sumitomo Pharma Osmotic Pumps, Durect, Cupertino, CA, USA) containing Co, Ltd, Osaka, Japan) is a nonselective α/β-adrenergic either arotinolol (0.25 mg · kg–1 · day–1, n=6) or 0.9% saline receptor blocker lacking local anesthetic, membrane-stabi- (n=6) was implanted randomly under anesthesia induced by lizing or intrinsic sympathomimetic properties, which means intraperitoneal injection of sodium pentobarbital (30 mg/kg). that arotinolol is a pureα /β-adrenergic receptor blocker.2,3 A previous study showed intraperitoneal administration of arotinolol has almost the same effects as intravenous administration.6 We did preliminary experiments to decide the dose of arotinolol required to reduce blood pressure (BP)

Received March 28, 2009; revised manuscript received August 12, 2009; accepted August 17, 2009; released online October 13, 2009 Department of Internal Medicine, *Intensive and Cardiac Care Unit, Nippon Medical School, Tokyo, Japan Mailing address: Naoki Sato, MD, Intensive and Cardiac Care Unit, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. E-mail: [email protected] All rights are reserved to the Japanese Circulation Society. For permissions, please e-mail: [email protected] Advance Publication by J-STAGE ISHIKAWA M et al.

Table. Effects of Arotinolol on Hemodynamics n Heart rate LVSP LVEDP Mean BP Mean RAP RVSP RVEDP (beats/min) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg) (mmHg)

Control 6 441±6 131±5 19±2 122±6 18±1 91±14 18±1 Arotinolol 6 432±15 132±6 15±1 115±11 12±2* 41±3* 11±1†

Values are mean ± standard error of mean. *P<0.05, †P<0.01. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; BP, blood pressure; RAP, right atrial pressure; RSVP, right ventricular systolic pressure; RVEDP, right ventricular end-diastolic pressure.

Figure 1. Representative waveforms of pulmonary arterial pressure (PAP) and right ventricular pressure (RVP) in control (Right) and arotinolol-treated (Left) rats.

Figure 2. Systolic and diastolic pulmonary arterial pressures (sPAP and dPAP, respectively) and mean PAP (mPAP) are shown. (Closed boxes) Arotinolol group, (open boxes) control group. In the arotinolol group, sPAP, dPAP, and mPAP are significantly lower compared with the control values. *P<0.05, †P<0.01. within less than 10% from baseline, and it was 0.25 mg · Koden Corporation, Tokyo, Japan) for measurement of pul- kg–1 · day–1, which we used in the present study. All rats monary hemodynamics, heart rate (HR), arterial pressure were kept in a clean room and allowed water and food. (AP), pulmonary artery pressure (PAP), and right ventricu- Two weeks after administration of MCT, rats were anesthe- lar pressure (RVP). The aortic pressure and left ventricular tized by intraperitoneal injection of sodium pentobarbital pressure (LVP) were measured via the carotid artery. End- (50 mg/kg) and a 1.4Fr Millar catheter (Millar Instruments, diastole was defined as the initial point of positive dP/dt. Houston, TX, USA) was inserted into the RV and pulmo- After completing these measurements, the rats were killed nary artery via the right jugular vein and right atrium (RA). and their hearts were removed immediately. Each heart was The catheter was connected to a pressure transducer (Nihon cut into 2 parts (right ventricular free wall [RV] and left Advance Publication by J-STAGE α /β-Blocker and PAH

Figure 3. Representative images of the right ventricles (RV). (Panel A) Cross-sections from control (Left) and arotinolol- treated (Right) rats. RV hypertrophy was less in the arotinolol-treated rats. (Panel B) Representative Masson-trichrome stained samples in normal (Left), control (Middle), and arotinolol-treated (Right) rats, showing that myocytes in the RV were the same size as those of a normal rat and different from those of the control rat. ventricle plus septum [LV+S]) according to the method of Statistical Analysis Fulton et al,7 and each part was weighted. The ratio of the All data are expressed as the mean ± standard error of mean. weight of either ventricle to the body weight (BW) was The effects of arotinolol were assessed by comparison with calculated as an index of LVH or RVH. saline using Student’s t-test. A P value <0.05 was considered to indicate statistical significance. All statistical analyses Myocyte Preparation and Measurement were performed with the Statistical Package for Social In several samples, cross-sectional specimens were im- Sciences version 11.0 (SPSS Inc, Chicago, IL, USA). mersed in 10% solution of formaldehyde. Masson-trichrome staining was performed to create representative images from both groups. To confirm precisely any differences in Results the degree of hypertrophy between the 2 groups, we exam- Hemodynamic Parameters (Table, Figures 1,2) ined the size of myocytes from the RV. After the physio- The number of rats in each group was six. There were no logical experiments, the hearts were rapidly removed and significant differences in HR, systolic LVP, or mean AP perfused with oxygenated (95% O2–5% CO2) calcium-free between the 2 groups. LVEDP was higher in the control Tyrode’s solution (mmol/L: NaCl 130, KCl 4.8, MgCl2 1.2, group, but the difference was not significant. Mean RAP HEPES 10, glucose 5; pH=7.4) at 37°C and then with and systolic RVP were significantly (P<0.05) higher in the Enzyme 1 Solution (type 2 collagenase 33 mg, hyaluroni- control group compared with the arotinolol group. The dase 21 mg, Tyrode’s solution 100 ml; Sigma Chemical Co, differences in RVEDP between groups were similar to St Louis, MO, USA). Next, the RV was removed from each those for mean RAP. In the arotinolol group, systolic PAP, heart and minced with scissors in a separate vessel con- diastolic PAP, and mean PAP were 32±2, 18±2, and 21± taining Enzyme 2 Solution (trypsin 20 mg and Enzyme 1 2 mmHg, respectively, which were significantly lower Solution 20 ml; Sigma). The tissue pieces were bubbled values than in the control group (73±8, 43±9, and 53± with 95% O2–5% CO2 for 10 min, filtered thorough a nylon 9 mmHg, respectively). mesh (300μ m), and washed with 20 ml of washing solution (Tyrode’s solution 15 ml and experimental solution 15 ml). Evaluation of Ventricular Hypertrophy (Figures 3,4) The tissue pieces were centrifuged at 550 rpm for 3 min Representative images of the cross-sectional specimens at room temperature. The supernatant was discarded and and histology are shown in Figure 3. Body weight was not the cells were resuspended on top of an albumin solution significantly different between the 2 groups. There were no (bovine serum albumin 1.2 g and Tyrode’s solution 20 ml). significant differences in LV+S/BW between the groups, Isolated myocytes were placed in a cell chamber on the but the RV/BW ratio of the arotinolol group was signifi- stage of an inverted microscope (Olympus, IX-70) that was cantly lower than that of the control group (0.58±0.01 vs continuously superfused at 37°C with the experimental 0.77±0.04 mg/g, P<0.01), suggesting that RVH was signifi- solution (mmol/L: 130 NaCl, 4.8 KCl, 1.2 MgCl2, 5 glucose, cantly prevented by arotinolol. Arotinolol also significantly 10 HEPES, 1 CaCl2). Cell length was measured using a prevented the increase in the maximum short-axis length SoftEdge video-based edge-detection system (IonOptix of myocytes to 24.7±0.8μ m vs 30.9±1.1μ m in the control Corporation, Milton, MA, USA). group (P<0.01), but not the maximum long-axis length Advance Publication by J-STAGE ISHIKAWA M et al.

Figure 4. Left ventricular (LV)/body weight (BW) ratio (Left) and right ventricular (RV)/ BW ratio (Right). There was no significant difference between groups in the LV/BW ratio. The RV/BW ratio was significantly lower in the arotinolol group. (Closed boxes) Arotinolol group, (open boxes) control group, respectively. NS, not significant.† P<0.01.

(113.9±2.9 vs 154.4±36.5μ m). activity might be related to the progression of RVH.13,14 In patients with isolated RV failure because of primary PAH, Bristow et al found selective β1-receptor downregulation Discussion and decreased basal adenylyl cyclase activity.15 Nervous The present study demonstrated that PAH and RVH could stimulation of the ventricles and abnormalities of the adren- be prevented by a pure α/β-adrenergic receptor blocker, ergic system are involved, because cardiac denervation pre- arotinolol, without a decrease in systemic BP, in a rat model vents the progression of sympathetic abnormalities and of MCT-induced PAH, suggesting that the α/β-adrenergic cardiac dysfunction.16 In the present study, we could not receptor blocker attenuated the progression of PAH and clarify the mechanism leading to the prevention of RVH, also prevented the development of RVH. but it is possible that early inhibition of the progression of In MCT-induced PAH, PA remodeling reportedly occurs, PAH might have delayed progression of RVH and inhibi- which also occurs in humans with primary PAH. Endothe- tion of cardiac sympathetic activity may have also had a lial dysfunction leads to a chronic decrease in the produc- direct preventive effect. tion of vasodilators, such as nitric oxide and prostacyclin, Another possibility for the prevention of the progression together with overexpression of vasoconstrictors, such as of PAH could be the indirect antiplatelet activity of α/β- ET-1 and thromboxanes, and also Rho-kinase activation.8,9 adrenergic receptor blockers. Platelet activation increases This imbalance induces vasoconstriction and smooth muscle the release of platelet-derived growth factor and serotonin cell activation, which leads to dysfunction, hyperplasia, and into the circulation. Increased availability of thromboxane, hypertrophy. In addition, there is inhibition of apoptosis, fibrinopeptide A, and plasminogen activator inhibitor-1 increased fibroblast proliferation, increased collagen depo- creates a pro-coagulant state in the pulmonary circulation, sition, and activation of pro-inflammatory cytokines and after which vasoconstriction, cell proliferation, and throm- angiogenesis. These abnormalities cause increased vascular bosis combine to cause deleterious pulmonary vascular tone and promote vascular remodeling.10 The role of the remodeling. The β-adrenergic receptor blockers have an sympathetic nervous system in the process of PA remodel- inhibitory effect on platelet function.17 Therefore, the indi- ing is not well known. Inoue et al demonstrated that rect antiplatelet action of β-adrenergic receptor blockers bunazosin hydrochloride, an α-adrenergic blocker, could might be a mechanism, but this should be confirmed by attenuate the elevation of RV systolic pressure, but not further studies. RVH, in the same animal model.11 Fujio et al demonstrated One more possibility for the prevention of PAH is direct that carvedilol or propranolol, but not prazosin, inhibited the inhibition of the effects of MCT by arotinolol. It is known proliferation of arterial smooth muscle cells from patients that MCT is metabolized in the liver and changed into with idiopathic PAH.5 Kanemoto et al reported patients pyrrole derivatives, which are toxic and cause vascular endo- with primary PAH whose condition improved markedly thelial and smooth cell proliferation, obliteration of arteri- when they received vasodilators combined with prazosin oles in the lung and induce RV hypertrophy with increased and isosorbide dinitrate.12 Thus, an α/β-adrenergic receptor RV pressure.18 Therefore, it is possible that arotinolol can blocker might improve PAH under appropriate conditions, directly inhibit the effects of all pyrrole derivatives of MCT but we do not know exactly which conditions are required immediately after administration. for the prevention of PAH. The present study results suggest that early pharmacological intervention with an α/β-adren- Study Limitations ergic receptor blocker might prevent progression of PAH, The present study did not directly examine pulmonary arte- although we did not directly examine endothelial function rial wall hypertrophy, but we expect that PA remodeling or histological abnormalities of the pulmonary arteries. would have been reduced in the arotinolol-treated rats Several previous studies using the MCT-induced PAH because of the significant prevention of PAH. The number model have shown downregulation of β1-adrenergic recep- of cardiacα - andβ -adrenergic receptors differs between rats tors and also a reduction of Gs protein-adenylyl cyclase and humans,19,20 and such differences could have influenced activity in the RV during progression of MCT-induced RVH the results of the present study. Therefore, we also need to and RV failure, which suggests that increased sympathetic examine the preventive effect of arotinolol in PAH patients. Advance Publication by J-STAGE α /β-Blocker and PAH

We examined only a 2-week treatment period in the monary arterial hypertension. Circ J 2006; 70: 174 – 178. present study. It is possible that arotinolol treatment delayed 9. Do.e Z, Fukumoto Y, Takaki A, Tawara S, Ohashi J, Nakano M, et al. the onset of PAH rather than attenuated the disease. In the Evidence for rho-kinase activation in patients with pulmonary arterial hypertension. Circ J 2009; 73: 1731 – 1739. present study, the aim was to reveal hemodynamic changes 10. Humbert M, Morrell NW, Archer SL, Stenmark KR, MacLean MR, during the development of PAH and RVH. It is reported Lang IM, et al. Cellular and molecular pathobiology of pulmonary that carvedilol improves survival rate in MCT-induced arterial hypertension. J Am Coll Cardiol 2004; 43(Suppl S): 13S – PAH rats.21 Therefore, we need to perform further study to 24S. 11. Inoue M, Watanabe K, Mori C, Tanaka O. The effect of bunazosin clarify the effect of arotinolol on the long-term outcome. on MCT-induced pulmonary hypertension in rats. Acta Paediatr Jpn We performed the experiments under anesthesia with 1994; 36: 133 – 138. pentobarbital, which reportedly elicits autonomic functions 12. Kanemoto N, Yamaguchi H, Shiina Y, Goto Y. Reversibility of in rats.22 Therefore, our data would be influenced by pento- primary pulmonary hypertension with vasodilator, anticoagulant and nocturnal oxygen therapy. Jpn Heart J 1989; 30: 929 – 934. barbital. However, differences between the 2 groups would 13. Ishikawa S, Honda M, Yamada S, Morioka S, Moriyama K. Biven- still be manifested even under these conditions and it is tricular down-regulation of beta-adrenergic receptors in right ven- extremely difficult to measure in conscious rats all the tricular hypertrophy induced by monocrotaline. Jpn Circ J 1991; 55: parameters investigated in the present study. 1077 – 1085. 14. Seyfarth T, Gerbershagen HP, Giessler C, Leineweber K, Heinroth- In conclusion, the present study demonstrated that the Hoffmann I, Ponicke K, et al. The cardiac beta-adrenoceptor-G- pure α/β-adrenergic receptor blocker, arotinolol, could protein(s)-adenylyl cyclase system in monocrotaline-treated rats. prevent the progression of MCT-induced PAH and RVH in J Mol Cell Cardiol 2000; 32: 2315 – 2326. rats, suggesting that sympathetic nervous activation plays 15. Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W, a role in the development of PAH. Rasmussen R, et al. Beta 1- and beta 2-adrenergic-receptor subpopu- lations in nonfailing and failing human ventricular myocardium: Cou- pling of both receptor subtypes to muscle contraction and selective References beta 1-receptor down-regulation in heart failure. Circ Res 1986; 59: 297 – 309. 1. Velez-Roa S, Ciarka A, Najem B, Vachiery JL, Naeije R, van de 16. Sato N, Vatner SF, Shen YT, Kudej RK, Ghaleh-Marzban B, Uechi Borne P. Increased sympathetic nerve activity in pulmonary artery M, et al. Effects of cardiac denervation on development of heart failure hypertension. Circulation 2004; 110: 1308 – 1312. and catecholamine desensitization. Circulation 1997; 95: 2130 – 2140. 2. Hara Y, Sato E. Pharmacological properties of S-596, a new beta- 17. Gasser JA, Betterridge DJ. Comparison of the effects of carvedilol, adrenergic blocking agent. Folia Pharmacol Jpn 1979; 75: 707 – 720. propranolol, and verapamil on in vitro platelet function in healthy 3. Miyagishi A, Nakahara H, Hara Y, Nakatani H. Effects of the new volunteers. J Cardiovasc Pharmacol 1991; 18(Suppl 4): S29 – S34. beta-adrenoceptor blocking agent, S-596 on the peripheral autonomic 18. Chang LT, Sun CK, Sheu JJ, Chiang CH, Youssef AA, Lee FY, et al. nervous system and smooth muscles. Arch Int Pharmacodyn Ther Cilostazol therapy attenuates monocrotaline-induced pulmonary arte- 1983; 261: 222 – 237. rial hypertension in rat model. Circ J 2008; 72: 825 – 831. 4. Watanabe H, Kakihana M, Ohtsuka S, Sugishita Y. Preventive effects 19. Castellano M, Böhm M. The cardiac beta-adrenoceptor-mediated of carvedilol on nitrate tolerance: A randomized, double-blind, signaling pathway and its alterations in hypertensive heart disease. placebo-controlled comparative study between carvedilol and arotin- Hypertension 1997; 29: 715 – 722. olol. J Am Coll Cardiol 1998; 32: 1201 – 1206. 20. Steinfath M, Chen YY, Lavicky J, Magnussen O, Nose M, Rosswag S, 5. Fujio H, Nakamura K, Matsubara H, Kusano KF, Miyaji K, Nagase et al. Cardiac alpha 1-adrenoceptor densities in different mammalian S, et al. Carvedilol inhibits proliferation of cultured pulmonary artery species. Br J Pharmacol 1992; 107: 185 – 188. smooth muscle cells of patients with idiopathic pulmonary arterial 21. Usui S, Yao A, Hatano M, Kohmoto O, Takahashi T, Nagai R, et al. hypertension. J Cardiovasc Pharmacol 2006; 47: 250 – 255. Upregulated neurohumoral factors are associated with left ventricular 6. Iba K, Mizuno K. Pharmacokinetic study of S-596 in mice and rats. remodeling and poor prognosis in rats with monocrotaline-induced Kiso to Rinsho 1987; 21: 4549 – 4579 (in Japanese). pulmonary arterial hypertension. Circ J 2006; 70: 1208 – 1215. 7. Fulton RM, Hutchinson EC, Jones AM. Ventricular weight in cardiac 22. Yang CC, Kuo TB, Chan SH. Auto- and cross-spectral analysis of hypertrophy. Br Heart J 1952; 14: 413 – 420. cardiovascular fluctuations during pentobarbital anesthesia in the rat. 8. Ishikura K, Yamada N, Ito M, Ota S, Nakamura M, Isaka N, et al. Am J Physiol 1996; 270(Heart Circ Physiol 39): H575 – H582. Beneficial acute effects of Rho-kinase inhibitor in patients with pul-